Evaluation of murine OX40L-murine IgG1(MM1) fusion protein on immunogenicity against L. mexicana infection in BALB/c mice.

The majority of OX40L is found on professional antigen-presenting cells (APC), the potency of OX40L to enhance the immunogenicity of potential vaccines against leishmania is not yet fully investigated. There is no report of administration of OX40L on cutaneous leishmaniasis either in therapy or prophylactic immunisation and the present study for the first time reports the effect of OX40L on L. mexicana infection. In this study, B9B8E2 cells were transfected with the murine OX40L and IgG1 plasmids, were used to produce the mOX40-mIgG1 (MM1). The therapeutic effects of MM1(mOX40L-mIgG1) was tested in a challenge experiment using L. mexicana infected BALB/c mice. Mice received two doses of MM1, on day 3 and 7 after the infection. Mice receiving MM1 generated an inflammatory reaction a few days after the injection of the OX40L, which was gradually dampened and finally disappeared 3 weeks later. There was a significant delay in the growth of developing lesions in mice receiving OX40L compared to controls injected with PBS and the size of lesions in the group receiving MM1 was significantly smaller than that of injected with either PBS. 40% of mice given MM1 remained lesion free for two months, when experiments were terminated. The results clearly indicate the high therapeutic effect of mOX40L-mIgG1 fusion protein in L. mexicana infection. The effect of OX40L on the enhancement of immunisation, needs to be further investigated for developing new vaccine strategies.

Inhibitory Effects of Leishmania Mexicana Infection on MHC-I Expression in Bone Marrow Derived Dendritic Cells.

Background: Leishmania is a parasite causing leishmaniasis with different clinical manifestations depending on the infectious species in many countries worldwide. Although different studies have been taken place to clear the interaction of the parasite with the immune system, many aspects of immunology of leishmaniasis is remained uncertain. Methods: Bone marrow derived dendritic cells (DCs) were cultured in vitro and divided into different groups (Nottingham Trent University, Nottingham, UK). The groups were separately infected with live or autoclaved L. mexicana or loaded with Soluble Leishmania Antigen (SLA). The expression of major histocompatibility complex class I (MHC-I) molecule was checked and compared on the cultured DCs using flow cytometry. Results: Infection of L. mexicana caused a significant downregulation in expression of molecules where killed Leishmania or SLA could not induce suppression in expression of these molecules. Conclusion: L. mexicana infection results in downregulation of MHC-I expression on bone marrow-derived dendritic cells.

Therapeutic effects of mesenchymal stem cells on cutaneous leishmaniasis lesions caused by Leishmania major.

OBJECTIVES: Leishmania major (L. major) is a cutaneous leishmaniasis causative agent. Current chemotherapeutic methods are not totally effective in treatment of this disease. The immunomodulation and tissue repairing capability of mesenchymal stem cells (MSCs), ease of isolation, detection and in vitro culture, have encouraged biologists to use MSCs for cell therapy in different infections such as cutaneous leishmaniasis. METHODS: BALB/c mice (6-8 weeks old) were infected with L. major then divided into four groups and treated with MSCs, Glucantime, Glucantime + MSCs, or PBS. Regression of lesions, potency of macrophages for phagocytosis, proliferation of immune cells against Leishmania soluble antigen, reduction of spleen parasite burden and healing of the lesions were evaluated on days 10, 20 and 30 of treatment. RESULTS: The results indicated that the mice intralesionally injected with MSCs showed significant regression in the lesions produced by L. major by day 30. Proliferation of splenocytes stimulated with SLA (soluble leishmania antigen) in vitro in MSC-treated mice on day 20 was significantly higher than in the other groups. The potency of phagocytosis in macrophages of mice treated with MSCs was significantly higher by day 30 and healing of the lesions in this group of mice showed more progress on histopathological examinations. Spleen parasite burden showed significant reduction in the mice treated with Glucantime + MSCs by day 30. CONCLUSIONS: The results showed that including MSCs in treatment of cutaneous leishmaniasis caused by L. major is a promising approach.

Leishmania Vaccines Entered in Clinical Trials: A Review of Literature.

Leishmaniasis is considered as a zoonotic infection and neglected tropical disease. Leishmania treatment is not totally successful and imposes high expenditures, especially in developing countries. Since the natural infection leads to the robust immunity in most of the human cases, many bodies of research have been focusing on Leishmania vaccines, being capable to control Leishmania infection. First generation vaccines (such as Leishmune® and CaniLeish®) have proved robust protective immunity in dogs. In human, recombinant vaccines, including Leish-F1 could confer some degrees of protective immunity against natural infection. Recently, ChAd63-KH DNA vaccine has been accomplished in providing prevention against Leishmania infection; however, this vaccine should be further evaluated in other clinical trials.

An investigation of the concurrency of anti-Neospora antibody and parasitemia in water buffalo (Bubalus bubalis) in northwest of Iran.

Neospora caninum is an obligate intracellular parasite causing abortion and reproductive failure in ruminants. Here, the seroprevalence of Neospora DNA and anti-Neospora antibodies and the correlation between the DNA and the antibody using polymerase chain reaction (PCR) and a new developed whole cell-based enzyme-linked immunosorbent assay (ELISA) in water buffalo (Bubalus bubalis) were investigated. To determine the level of anti-Neospora antibody, 83 serum samples were collected from buffaloes in the northwest of Iran. Plates were coated with 2 × 106 whole Neospora tachyzoites and the anti-Neospora antibody level was determined by calculating the ratio of sample/positive control (S/P) optical densities (ODs) in the ELISA. All samples with the ration of 0.50 or above were accounted as positive. To confirm the presence of Neospora DNA, the serum samples were directly subjected to PCR and nested PCR for detection of Neospora NC5 gene without the DNA isolation process. A total number of 83 buffalo serum samples were examined for the presence of anti-N. caninum immunoglobulin G and Neospora DNA. All samples with the S/P ratio of 0.50 or above (16 samples, 19.27%) were also positive for Neospora DNA. All samples with OD less than 0.50 (34 samples, 40.96%) were negative for Neospora DNA. However, 33 samples with the S/P ratio of bellow 0.50 (39.75%) showed a significant level of antibody. A 100% correlation was observed between high levels of the anti-Neospora antibody and Neospora DNA in the serum of water buffalo, and the whole N. caninum tachyzoites have the potency to be used as antigens for detection of the parasite in ELISA.

Potent in vitro antileishmanial activity of a nanoformulation of cisplatin with carbon nanotubes against Leishmania major.

OBJECTIVES: The aim of this study was to evaluate the cytotoxicity and antileishmanial activity of cisplatin-bonded carbon nanotubes both against promastigotes and amastigotes of Leishmania major in vitro. METHODS: Cisplatin-bonded single-walled carbon nanotubes (CP-SWCNT) and cisplatin-bonded multi-walled carbon nanotubes (CP-MWCNT) were considered as test compounds. In addition, SWCNT, MWCNT, free cisplatin and meglumine antimoniate (Glucantime®) were considered as controls. The effect of each compound was evaluated both on promastigote and amastigote stages of L. major and the results were compared. RESULTS: There was a statistically significant difference between the half-maximal inhibitory concentration (IC50) of CP-SWCNT and each of the controls, including SWCNT, cisplatin and Glucantime® (P<0.05). In addition, IC50 values of CP-MWCNT and each of the controls, including MWCNT, cisplatin and Glucantime®, were significantly different both for promastigotes and amastigotes (P<0.05). However, the selectivity index (SI) of CP-SWCNT was <10 (5.23), indicating that this compound is not completely safe. Moreover, the SI values of CP-MWCNT (12.54) and Glucantime® (16.28) were >10, indicating the selective effect of these two compounds on the parasite. Moreover, the IC50 of CP-MWCNT (0.11±0.09µM) for amastigotes was 41-fold lower than that of Glucantime® (4.52±1.31µM), suggesting that a lower dose of CP-MWCNT in comparison with Glucantime® is required to kill 50% of amastigotes. CONCLUSIONS: According to the potent in vitro antileishmanial activity of CP-MWCNT at low concentration against L. major, we suggest that they are evaluated in an animal model.

Expression of pro-inflammatory genes in lesions, spleens and blood neutrophils after burn injuries in mice treated with silver sulfodiazine.

OBJECTIVES: It is now supposed that cytokines released during the burn injuries have a great impact on the immunological and pathological responses after the burn. The main objective of this study was to investigate the expression of some pro-inflammatory genes in the wound, spleen and blood neutrophils during the healing process of burn wounds in a murine model. MATERIALS AND METHODS: The expression of ten pro-inflammatory genes were examined in wounds, spleens and blood neutrophils of mice with burn injuries treated with either silver sulfodiazine or phosphate-buffered saline (PBS) using RT-PCR at the end of the first and second weeks after injuries. RESULTS: None of the pro-inflammatory genes were expressed in the skin, spleen and blood neutrophils of healthy mice. In the group control, IL-12P35, IL-12P40, CCR5, IL-1ß and IFN-γ were expressed in the spleen and blood neutrophils in the first week. Instead, CCL5, CCR5, IL-1ß and IFN-γ were expressed in the wound, but in the second week, the expression of the genes became similar. In the test group, in the first week, TNF-α, IL-12P35, IL-12P40 and IL-1ß were expressed in the lesions, CCL4, IL-1α, IL-12P35, IL-12P40, CCR5 and IFN-γ were expressed in the spleen and no pro-inflammatory gene expression was detected in blood neutrophils. CONCLUSION: IL-1ß and IFN-γ are expressed in wound, spleen and neutrophils of untreated mice, but not in silver sulfodiazine treated mice. Hence, treatment with silver sulfodiazine suppressed the expression of pro-inflammatory genes in some stages of healing.

Evaluation of IL-12RB1, IL-12B, CXCR-3 and IL-17a expression in cases affected by a non-healing form of cutaneous leishmaniasis: an observational study design.

INTRODUCTION: Seldom cutaneous leishmaniasis (CL) may present as a lasting and active lesion(s), known as a non-healing form of CL (NHCL). Non-functional type 1 T helper (Th1) cells are assumed the most important factor in the outcome of the disease. The present study aims to assess some molecular defects that potentially contribute to Th1 impairment in NHCL. METHODS AND ANALYSIS: This prospective observational study will be implemented among five groups. The first and second groups comprise patients afflicted with non-healing and healing forms of CL, respectively. The third group consists of those recovered participants who have scars as a result of CL. Those participants who have never lived or travelled to endemic areas of leishmaniasis will comprise the fourth group. The fifth group comprises participants living in hyperendemic areas for leishmaniasis, although none of them have been afflicted by CL. The aim is to recruit 10 NHCL cases and 30 participants in each of the other groups. A leishmanin skin test (LST) will be performed to assess in vivo immunity against the Leishmania infection. The cytokine profile (interleukin (IL)-12p70, interferon (IFN)-γ, C-X-C motif chemokine ligand (CXCL)-11 and IL-17a) of the isolated peripheral blood mononuclear cells (PBMCs) will be evaluated through ELISA. Real-time PCR will determine the C-X-C motif chemokine receptor (CXCR)-3 and IL-17a gene expression and expression of IL-12Rß1 will be assessed by flow cytometry. Furthermore, IL-12B and IL-12RB1 mutation analysis will be performed. DISCUSSION: It is anticipated that the outcome of the current study will identify IL-12B and IL-12RB1 mutations, which lead to persistent lesions of CL. Furthermore, our expected results will reveal an association between NHCL and pro-inflammatory cytokines (IL-12p70, IFN-γ IL-17a and CXCL-11), as well as CXCR-3 expression. ETHICS AND DISSEMINATION: This study has been approved by a local ethical committee. The final results will be disseminated through peer-reviewed journals and scientific conferences.

Expression of Pro-inflammatory Genes in Lesions and Neutrophils during Leishmania major Infection in BALB/c Mice.

BACKGROUND: Leishmaniasis is a worldwide disease prevalent in tropical and sub-tropical countries in the world. Characterization of inflammatory responses produced in cutaneous leishmaniasis has not yet been completed. METHODS: The specific primers were designed for ten pro-inflammatory genes including CCL4, CCL3, TNF-α, IL-1α, IL-12P35, IL-12P40, CCL5, CCR5, IL-1ß and IFN- γ and their expression were assessed and compared using RT-PCR in the lesion and peripheral blood neutrophils in Leishmania infected BALB/c mice. RESULTS: None of the pro-inflammatory genes was expressed in the healthy tissue and except IFN-γ others were down-regulated by the parasite in the lesion in untreated mice. In mice treated with anti-Leishmanial drugs, the expression of the pro-inflammatory genes restarted. The figure of pro-inflammatory gene expression in neutrophils was different was from the lesions in treated and untreated mice. CONCLUSION: Leishmania is capable to suppress the expression of pro-inflammatory genes in the lesions but not in neutrophils. The expression of TNF-α in the lesions and down-regulation of IL-1ß in neutrophils could be accounted as an indication for healing of cutaneous leishmaniasis. The results open a new window on characterization of Leishmania lesions and clarifying the role of neutrophils in Leishmania infections.

An overview on Leishmania vaccines: A narrative review article.

Leishmaniasis is one of the major health problems and categorized as a class I disease (emerging and uncontrolled) by World Health Organization (WHO), causing highly significant morbidity and mortality. Indeed, more than 350 million individuals are at risk of Leishmania infection, and about 1.6 million new cases occur causing more than 50 thousands death annually. Because of the severe toxicity and drug resistance, present chemotherapy regimen against diverse forms of Leishmania infections is not totally worthwhile. However, sound immunity due to natural infection, implies that vigor cellular immunity against Leishmania parasites, via their live, attenuated or killed forms, can be developed in dogs and humans. Moreover, genetically conserved antigens (in most of Leishmania species), and components of sand fly saliva confer potential immunogenic molecules for Leishmania vaccination. Vaccines successes in animal studies and some clinical trials clearly justify more researches and investments illuminating opportunities in suitable vaccine designation.

CTL responses to DCs stimulated with leishmania antigens detected by DCs expressing Leishmania gp63.

BACKGROUND: Leishmania is a pathogenic parasite which infects mononuclear cells in vertebrate hosts. Different strategies have been taken to develop immunity against Leishmania. DCs loaded with immunogenic antigen have resulted in different levels of Th1-type immune response and cytotoxic T lymphocytes (CTL) activity. OBJECTIVE: To evaluate the potency of DCs primed with soluble Leishmania mexicana antigens (SLA) in developing CTL activity. METHODS: DCs were loaded with SLA and injected to Balb/c mice. After two weeks the mice were sacrificed and their splenocytes were used as effector cells in a standard 4-hour cytotoxicity assay against DCs transfected with pcDNA3 containing L. mexicana gp63 gene. RESULTS: Immunization of Balb/c mice with DCs loaded with SLA resulted in high levels of CTL activity against DCs transfected with pcDNA3 containing L. mexicana gp63 gene. CONCLUSIONS: The results indicate a high potency for DCs primed with Leishmania antigens in inducing CTL activity, which can be used for developing an immunogenic vaccine against Leishmania.